Malaria is a major health problem worldwide, with over 300 million people becoming infected and up to one million deaths annually. The emergence and spread of resistance to most anti-malarial drugs has made the effective treatment of malaria difficult and there is an urgent need for new anti-malarial drugs or drug combinations. Atovaquone is a safe, effective drug that is used in combination with proguanil for the treatment and prevention of malaria. Previous studies have shown that clinical resistance to atovaquone is conferred by single nucleotide polymorphisms (SNPs) in the mitochondrial encoded cytochrome b gene of P. falciparum. Although multiple non-synonymous SNPs can be selected under drug pressure in vitro, thus far clinical resistance is limited to amino acid substitutions at position 268 (e.g., Y268S). From phase II studies of atovaquone in Thailand, we have characterized multiple isolates of P. falciparum that were collected from patients that failed treatment with atovaquone alone (various dose regimens) or in combination with either proguanil or pyrimethamine. Interestingly, we observed a broad range of resistance to atovaquone, from 5 to >10,000 fold in these isolates. The low-grade resistant isolates possessed no cytochrome b mutations, whereas the moderate to extreme resistant isolates all had aa268 mutations. Given the broad range of resistance observed, more than just the Y268S mutation seemed likely to be the sole basis for resistance. By using a series of inhibitors we found extremely atovaquone resistant parasites that are highly resistant to all drugs tested that target the mitochondria. These include the normally potent 4(1H)-quinolones and pyridones, and dihydroorotate dehydrogenase (DHODH) inhibitors. The major goals of this project are to identify the molecular markers of resistance, that along with cytochrome b mutations convey an extreme resistance phenotype. We will clone P. falciparum from admission and recrudescence isolates from the atovaquone Phase 2 clinical studies to obtain reference clones with low, moderate, and extreme resistance to mitochondrial inhibitors. We will use next generation sequencing to identify novel SNPs or CNVs associated with resistance; this effort will be aided by comparing the resistant clones with P. falciparum clones from the same patient prior to treatment. In addition, we will test the hypothesis that extreme resistance was selected in vivo by simultaneous exposure to atovaquone and pyrimethamine. Finally we will assess the potential for transmission of resistance genotypes to mosquitos. The results of these studies will provide critical knowledge about the resistance risks and potential for spread of extreme resistance to mitochondrial inhibitors in the field.